Bacillus sphaericus is a sporeforming bacterium with a high degree of insecticidal activity toward larvae of certain mosquito species. This bacterium is being developed as a microbial control agent for use in integrated vector control programs. Among the nearly 40 insecticidal strains of B. sphaericus currently known two very similar strains, 1593 and 2362, appear to have the greatest potential utility. Bacillus sphaericus 1593 kills mosquito larvae by means of a protein toxin of ca. 50 Kdal, which is produced during sporulation. This toxin is packaged in a parasporal crystal and to a lesser extent in the spore itself. Intoxicated larvae show gut and behavioral symptoms within 1 hr of ingestion of large dose of B. sphaericus, and may die in as little as 4 hr. The objectives of this proposal are to develop an invitro assay for the B. sphaericus toxin utilizing cultured mosquito cells and to use this assay to investigate parameters of the host-pathogen interaction including toxin activation, binding, and molecular mode of action, as well as ultrastructural changes in the cells. Toxin will be purified using a combination of gel and ion exchange chromatography, possibly followed by affinity or HPLC chromatography. Monoclonal and polyclonal antibodies raised to the toxin will be utilized in purification and localization of the toxin.
